Mycoplasma pneumoniae, a human pathogen, primarily causes pneumonia and various other respiratory infections and contributes significantly to worldwide morbidity and mortality. The clinical features associated with M. pneumoniae are often indistinguishable from a variety of other viral and atypical bacterial pathogens, and infections tend to be quite chronic and transmit fast in community-populations. Rapid detection of the microbial agent with high sensitivity and specificity is critical for appropriate treatment and control of disease transmission. The specific focus of this project is to develop a rapid detection method that requires fewer steps to completion, is simple to adopt in a clinical setting, and yet will provide high sensitivity and specificity. The method will be based on single-step amplification of a gene specific of M. pneuminiae in the presence of a stem-loop structured fluorescent energy transfer probe. The amplification will be by a DNA polymerase potentially insensitive to specimen-inhibitor so as to reduce the limitation as applied in clinical samples. The probe will be designed to hybridize specifically with its target; and only upon probe-target hybrid formation, fluorescence will be emitted from a reporter fluorophore attached to it. The presence of M. pneumoniae will be detected by fluorescence measurement of a post-nucleic acid amplified sample immediately after amplification, thereby requiring no additional steps. The method will also be tested in prototype strains and clinical isolates of M. pneumoniae to examine the feasibility of applying it in actual specimens to be tested in Phase II.